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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 1,495 records · Page 83
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Mycobacterium tuberculosis Phe-tRNA synthetase: structural insights into tRNA recognition and aminoacylation

Abstract Tuberculosis, caused by Mycobacterium tuberculosis, responsible for ∼1.5 million fatalities in 2018, is the deadliest infectious disease. Global spread of multidrug resistant strains is a public health threat, requiring new treatments. Aminoacyl-tRNA synthetases are plausible candidates as potential drug targets, because they play an essential role in translating the DNA code into protein sequence by attaching a specific amino acid to their cognate tRNAs. We report structures of M. tuberculosis Phe-tRNA synthetase complexed with an unmodified tRNAPhe transcript and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. High-resolution models reveal details of two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3′ end of tRNAPhe with the adenylate site. For the first time, we observe the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism. We biochemically validated the inhibitory potency of the adenylate analog and provide the most complete view of the Phe-tRNA synthetase/tRNAPhe system to date. The presented topography of amino adenylate-binding and editing sites at different stages of tRNA binding to the enzyme provide insights for the rational design of anti-tuberculosis drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

ClusterCAD 2.0: an updated computational platform for chimeric type I polyketide synthase and nonribosomal peptide synthetase design

Abstract Megasynthase enzymes such as type I modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) play a central role in microbial chemical warfare because they can evolve rapidly by shuffling parts (catalytic domains) to produce novel chemicals. If we can understand the design rules to reshuffle these parts, PKSs and NRPSs will provide a systematic and modular way to synthesize millions of molecules including pharmaceuticals, biomaterials, and biofuels. However, PKS and NRPS engineering remains difficult due to a limited understanding of the determinants of PKS and NRPS fold and function. We developed ClusterCAD to streamline and simplify the process of designing and testing engineered PKS variants. Here, we present the highly improved ClusterCAD 2.0 release, available at https://clustercad.jbei.org. ClusterCAD 2.0 boasts support for PKS-NRPS hybrid and NRPS clusters in addition to PKS clusters; a vastly enlarged database of curated PKS, PKS-NRPS hybrid, and NRPS clusters; a diverse set of chemical ‘starters’ and loading modules; the new Domain Architecture Cluster Search Tool; and an offline Jupyter Notebook workspace, among other improvements. Together these features massively expand the chemical space that can be accessed by enzymes engineered with ClusterCAD.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Improved genome editing by an engineered CRISPR-Cas12a

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis - and trans -DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism

Abstract Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNAPyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Conserved unique peptide patterns (CUPP) online platform 2.0: implementation of +1000 JGI fungal genomes

Carbohydrate-processing enzymes, CAZymes, are classified into families based on sequence and three-dimensional fold. Because many CAZyme families contain members of diverse molecular function (different EC-numbers), sophisticated tools are required to further delineate these enzymes. Such delineation is provided by the peptide-based clustering method CUPP, Conserved Unique Peptide Patterns. CUPP operates synergistically with the CAZy family/subfamily categorizations to allow systematic exploration of CAZymes by defining small protein groups with shared sequence motifs. The updated CUPP library contains 21,930 of such motif groups including 3,842,628 proteins. The new implementation of the CUPP-webserver, https://cupp.info/, now includes all published fungal and algal genomes from the Joint Genome Institute (JGI), genome resources MycoCosm and PhycoCosm, dynamically subdivided into motif groups of CAZymes. This allows users to browse the JGI portals for specific predicted functions or specific protein families from genome sequences. Thus, a genome can be searched for proteins having specific characteristics. All JGI proteins have a hyperlink to a summary page which links to the predicted gene splicing including which regions have RNA support. The new CUPP implementation also includes an update of the annotation algorithm that uses only a fourth of the RAM while enabling multi-threading, providing an annotation speed below 1 ms/protein.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Perspectives on improving photosynthesis to increase crop yield

Abstract Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin–Benson–Bassham (CBB) cycle. Additionally, we explore methods to increase carbon dioxide (CO2) concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

p HBMT1, a BAHD-family monolignol acyltransferase, mediates lignin acylation in poplar

Abstract Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol–p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free translation system and found five enzymes capable of producing monolignol–p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol–p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol–p-hydroxybenzoates and cell-wall-bound monolignol–p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The intermediate in a nitrate-responsive ω-amidase pathway in plants may signal ammonium assimilation status

Abstract A metabolite of ammonium assimilation was previously theorized to be involved in the coordination of the overall nitrate response in plants. Here we show that 2-hydroxy-5-oxoproline, made by transamination of glutamine, the first product of ammonium assimilation, may be involved in signaling a plant’s ammonium assimilation status. In leaves, 2-hydroxy-5-oxoproline met four foundational requirements to be such a signal. First, when it was applied to foliage, enzyme activities of nitrate reduction and ammonium assimilation increased; the activities of key tricarboxylic acid cycle-associated enzymes that help to supply carbon skeletons for amino acid synthesis also increased. Second, its leaf pools increased as nitrate availability increased. Third, the pool size of its precursor, Gln, reflected ammonium assimilation rather than photorespiration. Fourth, it was widely conserved among monocots, dicots, legumes, and nonlegumes and in plants with C3 or C4 metabolism. Made directly from the first product of ammonium assimilation, 2-hydroxy-5-oxoproline acted as a nitrate uptake stimulant. When 2-hydroxy-5-oxoproline was provided to roots, the plant’s nitrate uptake rate approximately doubled. Plants exogenously provided with 2-hydroxy-5-oxoproline to either roots or leaves accumulated greater biomass. A model was constructed that included the proposed roles of 2-hydroxy-5-oxoproline as a signal molecule of ammonium assimilation status in leaves, as a stimulator of nitrate uptake by roots and nitrate downloading from the xylem. In summary, a glutamine metabolite made in the ω-amidase pathway stimulated nitrate uptake by roots and was likely to be a signal of ammonium assimilation status in leaves. A chemical synthesis method for 2-hydroxy-5-oxoproline was also developed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

SPOTTED-LEAF7 targets the gene encoding β-galactosidase9, which functions in rice growth and stress responses

Abstract β-Galactosidases (Bgals) remove terminal β-D-galactosyl residues from the nonreducing ends of β-D-galactosidases and oligosaccharides. Bgals are present in bacteria, fungi, animals, and plants and have various functions. Despite the many studies on the evolution of BGALs in plants, their functions remain obscure. Here, we identified rice (Oryza sativa) β-galactosidase9 (OsBGAL9) as a direct target of the heat stress-induced transcription factor SPOTTED-LEAF7 (OsSPL7), as demonstrated by protoplast transactivation analysis and yeast 1-hybrid and electrophoretic mobility shift assays. Knockout plants for OsBGAL9 (Osbgal9) showed short stature and growth retardation. Histochemical β-glucuronidase (GUS) analysis of transgenic lines harboring an OsBGAL9pro:GUS reporter construct revealed that OsBGAL9 is mainly expressed in internodes at the mature stage. OsBGAL9 expression was barely detectable in seedlings under normal conditions but increased in response to biotic and abiotic stresses. Ectopic expression of OsBGAL9 enhanced resistance to the rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, as well as tolerance to cold and heat stress, while Osbgal9 mutant plants showed the opposite phenotypes. OsBGAL9 localized to the cell wall, suggesting that OsBGAL9 and its plant putative orthologs likely evolved functions distinct from those of its closely related animal enzymes. Enzyme activity assays and analysis of the cell wall composition of OsBGAL9 overexpression and mutant plants indicated that OsBGAL9 has activity toward galactose residues of arabinogalactan proteins (AGPs). Our study clearly demonstrates a role for a member of the BGAL family in AGP processing during plant development and stress responses.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Arabidopsis ACYL CARRIER PROTEIN4 and RHOMBOID LIKE10 act independently in chloroplast phosphatidate synthesis

Abstract ACYL CARRIER PROTEIN4 (ACP4) is the most abundant ACP isoform in Arabidopsis (Arabidopsis thaliana) leaves and acts as a scaffold for de novo fatty acid biosynthesis and as a substrate for acyl-ACP-utilizing enzymes. Recently, ACP4 was found to interact with a protein-designated plastid RHOMBOID LIKE10 (RBL10) that affects chloroplast monogalactosyldiacylglycerol (MGDG) biosynthesis, but the cellular function of this interaction remains to be explored. Here, we generated and characterized acp4 rbl10 double mutants to explore whether ACP4 and RBL10 directly interact in influencing chloroplast lipid metabolism. Alterations in the content and molecular species of chloroplast lipids such as MGDG and phosphatidylglycerol were observed in the acp4 and rbl10 mutants, which are likely associated with the changes in the size and profiles of diacylglycerol (DAG), phosphatidic acid (PA), and acyl-ACP precursor pools. ACP4 contributed to the size and profile of the acyl-ACP pool and interacted with acyl-ACP-utilizing enzymes, as expected for its role in fatty acid biosynthesis and chloroplast lipid assembly. RBL10 appeared to be involved in the conversion of PA to DAG precursors for MGDG biosynthesis as evidenced by the increased 34:x PA and decreased 34:x DAG in the rbl10 mutant and the slow turnover of radiolabeled PA in isolated chloroplasts fed with [14C] acetate. Interestingly, the impaired PA turnover in rbl10 was partially reversed in the acp4 rbl10 double mutant. Collectively, this study shows that ACP4 and RBL10 affect chloroplast lipid biosynthesis by modulating substrate precursor pools and appear to act independently.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Modular cell-free expression plasmids to accelerate biological design in cells

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy to use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desirable metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes, and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has matured as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for Clostridium autoethanogenum expression with efficiencies ranging from 68% to 90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A Novel Zinc Binding Group for HDAC6 Inhibition

Histone deacetylases (HDAC’s) are key regulatory enzymes in gene transcription and cellular motility through the deacetylation of lysine residues. These enzymes bear a distinct clinical significance, as the upregulation of HDACs has been associated with oncogenesis for many hematological malignancies and proliferation of other neurodegenerative or immune disorders. There are four different classes of HDACs, three of which require zinc for catalysis. Among the zinc dependent isozymes, HDAC6 is thought to be a particularly desirable therapeutic target, as its selective inhibition in malignant cells is accompanied by fewer associated toxicities in comparison to pan‐HDAC inhibition. Therefore, optimizing HDAC6 inhibitor selectivity has the potential to yield great clinical significance. This selectivity is often conferred by designing inhibitors with bulky capping groups and a hydrophobic linker region, which make favorable interactions in the hydrophobic region of the HDAC6 active site. Many inhibitors contain a hydroxamate zinc‐binding group, which can coordinate with bidentate or monodentate geometry. In efforts to optimize HDAC6 selectivity by exploring alternative zinc‐binding groups, a unique inhibitor containing an oxadiazole ring was discovered. Surprisingly, the crystal structure of its complex with HDAC6 reveals that the oxadiazole undergoes a ring opening reaction to yield an acylhydrazide that binds with an extended conformation in the active site.

Cragin, Abigail↗
DOE OSTI2024

Steroid responsiveness in alcohol-associated hepatitis is linked to glucocorticoid metabolism, mitochondrial repair, and heat shock proteins

Alcohol-associated hepatitis (AH) is one of the clinical presentations of alcohol-associated liver disease. AH has poor prognosis, and corticosteroids remain the mainstay of drug therapy. However, ~40% of patients do not respond to this treatment, and the mechanisms underlying the altered response to corticosteroids are not understood. The current study aimed to identify changes in hepatic protein expression associated with responsiveness to corticosteroids and prognosis in patients with AH. Patients with AH were enrolled based on the National Institute on Alcohol Abuse and Alcoholism inclusion criteria for acute AH and further confirmed by a diagnostic liver biopsy. Proteomic analysis was conducted on liver samples acquired from patients with AH grouped as nonresponders (AH-NR, n = 7) and responders (AH-R, n = 14) to corticosteroids, and nonalcohol-associated liver disease controls (n = 10). The definition of responders was based on the clinical prognostic model, the Lille Score, where a score < 0.45 classified patients as AH-R and a score > 0.45 as AH-NR. Primary outcomes used to assess steroid response were Lille Score (eg, improved liver function) and survival at 24 weeks. Reduced levels of the glucocorticoid receptor and its transcriptional co-activator, glucocorticoid modulatory element-binding protein 2, were observed in the hepatic proteome of AH-NR versus AH-R. The corticosteroid metabolizing enzyme, 11-beta-hydroxysteroid dehydrogenase 1, was increased in AH-NR versus AH-R along with elevated mitochondrial DNA repair enzymes, while several proteins of the heat shock pathway were reduced. Analysis of differentially expressed proteins in AH-NR who survived 24 weeks relative to AH-NR nonsurvivors revealed several protein expression changes, including increased levels of acute phase proteins, elevated coagulation factors, and reduced mast cell markers. This study identified hepatic proteomic changes that may predict responsiveness to corticosteroids and mortality in patients with AH.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Association of human T-cell leukemia virus type 1 with prevalent rheumatoid arthritis among atomic bomb survivors: A cross-sectional study

Previous studies have suggested that human T-cell leukemia virus type 1 (HTLV-1) might act as a pathogen in rheumatoid arthritis (RA), but epidemiological evidence of an association is scarce. We measured anti-HTLV-1 antibodies among Nagasaki atomic bomb survivors to determine whether HTLV-1 is related to RA and whether radiation exposure is associated with HTLV-1 and RA prevalence. This is a cross-sectional study among atomic bomb survivors who participated in biennial health examinations from 2006 to 2010. Serum levels of anti-HTLV-1 antibodies were measured using a chemiluminescent enzyme immunoassay and confirmed by Western blotting. Association between HTLV-1 and RA was analyzed by a logistic regression model. Of 2091 participants (women 61.5%; median age, 73 years), 215 (10.3%) had anti-HTLV-1 antibodies. HTLV-1 prevalence was higher among women (13.1% vs 5.8%; P<.001). Twenty-two participants (1.1%) were diagnosed with RA. HTLV-1 prevalence among RA participants was significantly higher than that among non-RA participants (27.3% vs 10.1%; P=.020). After adjustment for age, sex, and hepatitis C virus infection, HTLV-1 was significantly associated with prevalent RA (odds ratio, 2.89; 95% confidence interval, 1.06, 7.03). There was no association between radiation dose and either the prevalence of HTLV-1 or RA. This study, among a well-defined group of atomic bomb survivors, suggests that HTLV-1 is associated with RA. Abbreviations: AHS = Adult Health Study, ATL/ATLL = adult T cell leukemia/lymphoma, bioDMARDs = biological disease modifying antirheumatic drugs, CCP = cyclic citrullinated peptide, CI = confidence interval, CLEIA = chemiluminescent enzyme immunoassay, CRP = C-reactive protein, csDMARDs = conventional synthetic disease modifying antirheumatic drugs, Gy = gray, HCV = hepatitis C virus, HTLV-1 = human T-cell leukemia virus type 1, IF = immunofluorescent, RA = rheumatoid arthritis, RERF = Radiation Effects Research Foundation, RF = rheumatoid factor, SS = Sjogren syndrome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

An HMM approach expands the landscape of sesquiterpene cyclases across the kingdom Fungi

Sesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii, each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Deciphering the altered conformational states of bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus

Abstract The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.

Destan, Ebru (ORCID:0000000231290827)↗
DOE OSTI2020

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Understanding the selectivity of nonsteroidal anti-inflammatory drugs for cyclooxygenases using quantum crystallography and electrostatic interaction energy

Quantum crystallography methods have been employed to investigate complex formation between nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX) enzymes, with particular focus on the COX-1 and COX-2 isoforms. This study analyzed the electrostatic interaction energies of selected NSAIDs (flurbiprofen, ibuprofen, meloxicam and celecoxib) with the active sites of COX-1 and COX-2, revealing significant differences in binding profiles. Flurbiprofen exhibited the strongest interactions with both COX-1 and COX-2, indicating its potent binding affinity. Celecoxib and meloxicam showed a preference for COX-2, consistent with their known selectivity for this isoform, while ibuprofen showed comparable interaction energies with both isoforms, reflecting its nonselective inhibition pattern. Key amino-acid residues, including Arg120, Arg/His513 and Tyr355, were identified as critical determinants of NSAID selectivity and binding affinity. The findings highlight the complex interplay between interaction energy and selectivity, suggesting that while electrostatic interactions play a fundamental role, additional factors such as enzyme dynamics and the hydrophobic effect also contribute to the therapeutic efficacy and safety profiles of NSAIDs. These insights provide valuable guidance for the rational design of NSAIDs with enhanced therapeutic benefits and minimized adverse effects.

60 APPLIED LIFE SCIENCES↗